Optimal amounts of ascorbic acid (vitamin C) for maintaining human health are unknown. As a unique means to address this problem we developed the concept of in situ kinetics, in which the principles of reaction kinetics are applied to vitamin C dependent reactions in situ. Studies of catecholamine biosynthesis are the first proof that kinetics in situ can be characterized. The experiments also provide new evidence that reaction mechanisms and kinetics are different for reactions in situ compared to isolated reactions, and indicate that it is essential to study kinetics in situ to determine vitamin requirements. We previously discovered that ascorbic acid is accumulated by human neutrophils in mM concentration. We recently found that the physiologic structural analog glucose exquisitely regulates ascorbic acid transport by two distinct mechanisms. We also discovered that ascorbate accumulation in neutrophils can be upregulated 10 fold. The mechanism is by extracellular oxidation of ascorbate to dehydroascorbic acid, preferential transmembrane translocation of dehydroascorbic acid, and immediate intracellular reduction to ascorbate. These experiments are the first demonstration that ascorbate recycling occurs physiologically. To learn vitamin C function in human lymphocytes, experiments using differential hybridization and subtraction cloning are underway. Ascorbic acid accumulation in human fibroblasts was characterized as a prelude to understanding how different concentrations of the vitamin regulate proline hydroxylation. Clinical goals are to learn how much vitamin C is found in humans as a function of ingestion, so that ascorbic acid dependent reactions can occur. An IRB approved inpatient clinical trial has been to learn for the first time how ascorbic acid ingestion regulates plasma and tissue concentrations in healthy humans. Five subjects who were inpatients for 4-6 months have completed the study and two additional subjects are inpatients. These data will provide new information and will permit human ascorbate requirements to be determined using in situ kinetics.